Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale, allowing us to unlock the mysteries of how biomolecules work together to achieve the complexity that is a cell. This high-resolution, non-destructive method for examining subcellular events has opened up an exciting new frontier: the study of macromolecular localization and dynamics in living cells. We have developed methods for single-molecule investigations inside live bacteria cells in order to investigate DNA mismatch repair, which corrects base-pairing errors in Bacillus subtilis. Based on Photoactivated Localization Microscopy (PALM), two-color single-particle tracking, and genetic mutations, we reveal an intimate and dynamic coupling between DNA mismatch recognition and DNA replication that is used by cells to maintain high-fidelity genome replication.Though these single-molecule tracking experiments permit nanometer-scale measurements with millisecond time resolution, in vivo super-resolution imaging is limited by dim and unstable fluorophores. We therefore take advantage of the localized surface plasmon resonances supported by metal nanoparticles. In particular, we have extended the implications of plasmon-enhanced fluorescence to the realm of live-cell bio-imaging by using biocompatible gold nanorods to improve the emissive properties of single intrinsically fluorescent proteins in vitro, and we have applied this coupling to single-molecule fluorescence imaging of sub-cellular protein dynamics in living bacteria. Overall, our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging, and that plasmon-enhanced imaging is a promising technique for improving live-cell single-molecule microscopy.

Increasing imaging resolution to reveal cooperativity and dynamics in live bacteria

248th National Meeting (2014)

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